Single molecule fluorescence techniques applied for PA-Nter activity measurements.
Histograms of single molecule FRET efficiency of 30-nt fluorescent hairpin DNA (A) before the reaction, (B) after the reaction with PA-Nter; E = 0 indicates no FRET, E = 1 indicates maximal FRET, S denotes photon stoichiometry. Example histogram shown from a series of three independent experiments. The correlation functions of hairpin DNA measured with FCCS, (C) maximum cross-correlation before the reaction, (D) minimum cross-correlation after the reaction with PA-Nter. Blue line indicates cross-correlation, green and red denote auto-correlation functions. (E) Cross-correlation functions in 0, 5, 10, 15, 20, 25 and 30 minute of the reaction of 15 nM hairpin DNA with 100 nM PA-Nter. Insert picture shows the concentration of the hairpin DNA (nM) throughout the reaction time. (F) Comparison with electrophoretic assay. Products of the cleavage of 5.4 μM hairpin DNA with 3.3 μM PA-Nter in the presence of 1 mM ions and 2 mM DTT. Samples were incubated at 25°C for 1 h and inactivated at 70°C for 5 min.